Metabolic elicitors of Pseudomonas fluorescens N 21.4 elicit flavonoid metabolism in blackberry fruit.

BACKGROUND: The beneficial rhizobacterium, Pseudomonas fluorescens N 21.4, and its metabolic elicitors were inoculated in commercial cultivars of blackberry plants (Rubus cv. Loch Ness). Phenolic compounds present in red and black fruit and the expression of structural marker genes of the phenylpropanoid pathway during fruit ripening were studied. RESULTS: An inverse relationship between gene expression and accumulation of metabolites was seen, except for the RuDFR gene, which had a direct correlation with cyanidin 3-O-glucoside synthesis, increasing its content 1.3 times when RuDFR was overexpressed in the red fruit of plants inoculated with the metabolic elicitors of P. fluorescens N 21.4, compared with red fruit of plants inoculated with N 21.4. The RuCHS gene also had a fundamental role in the accumulation of metabolites. Both rhizobacterium and metabolic elicitors triggered the flavonoid metabolism, enhancing the catechin and epicatechin content between 1.1 and 1.6 times in the case of red fruit and between 1.1 and 1.8 times in the case of black fruit. Both treatments also boosted the anthocyanin, quercetin, and kaempferol derivative content, highlighting the effects of metabolic elicitors in red fruit and the effects of live rhizobacterium in black fruit. CONCLUSION: The metabolic elicitors' capacity to modulate gene expression and to increase secondary metabolites content was demonstrated. This work therefore suggests that they are effective, affordable, easily manageable, and ecofriendly plant inoculants that complement, or are alternatives to, beneficial rhizobacteria. © 2020 Society of Chemical Industry.

Biotic elicitation as a tool to improve strawberry and raspberry extract potential on metabolic syndrome-related enzymes in vitro.

BACKGROUND: Raspberry and strawberry are high value-added food products that can contribute to human health due to the abundance of polyphenols that they contain. Polyphenols are secondary metabolites and therefore devoted to improve plant adaptation, these polyphenol profile can be induced applying different stimuli, such as certain bacteria. The aim of this study was twofold: (i) to evaluate the ability of two bacterial strains to modulate secondary metabolisms in strawberry and raspberry, and (ii) to explore the ability of plant extracts to modify enzyme activities related to metabolic syndrome. RESULTS: Total phenolic and anthocyanin content was higher in strawberries than in raspberries, despite similar antioxidant capacities. Strawberry extracts performed better on the tested enzymes, except on α-glucosidase inhibition capacity. Bacillus amyloliquefaciens stabilized the effects of extracts at different points in time, and Pseudomonas fluorescens modified plant metabolism after more inoculations (spring) in both species, improving the effects of raspberry extracts on α-glucosidase, COX1, and COX2, and of strawberry on α-amylase and COX1. CONCLUSION: It is good to include these two fruits in the diet because they improve the activity of metabolic syndrome-related enzymes. Applying either strain during plant growth modifies the bioactive profile of the plants, improving the effects of the fruit extracts on human health. © 2018 Society of Chemical Industry.

RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. Var. Lochness) fruit.

BACKGROUND: There is an increasing interest in berries, especially blackberries in the diet, because of recent reports of their health benefits due to their high content of flavonoids. A broad range of genomic tools are available for other Rosaceae species but these tools are still lacking in the Rubus genus, thus limiting gene discovery and the breeding of improved varieties. RESULTS: De novo RNA-seq of ripe blackberries grown under field conditions was performed using Illumina Hiseq 2000. Almost 9 billion nucleotide bases were sequenced in total. Following assembly, 42,062 consensus sequences were detected. For functional annotation, 33,040 (NR), 32,762 (NT), 21,932 (Swiss-Prot), 20,134 (KEGG), 13,676 (COG), 24,168 (GO) consensus sequences were annotated using different databases; in total 34,552 annotated sequences were identified. For protein prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 32,540. Non redundant (NR), annotation showed that 25,418 genes (73.5%) has the highest similarity with Fragaria vesca subspecies vesca. Reanalysis was undertaken by aligning the reads with this reference genome for a deeper analysis of the transcriptome. We demonstrated that de novo assembly, using Trinity and later annotation with Blast using different databases, were complementary to alignment to the reference sequence using SOAPaligner/SOAP2. The Fragaria reference genome belongs to a species in the same family as blackberry (Rosaceae) but to a different genus. Since blackberries are tetraploids, the possibility of artefactual gene chimeras resulting from mis-assembly was tested with one of the genes sequenced by RNAseq, Chalcone Synthase (CHS). cDNAs encoding this protein were cloned and sequenced. Primers designed to the assembled sequences accurately distinguished different contigs, at least for chalcone synthase genes. CONCLUSIONS: We prepared and analysed transcriptome data from ripe blackberries, for which prior genomic information was limited. This new sequence information will improve the knowledge of this important and healthy fruit, providing an invaluable new tool for biological research.

Supplementing diet with blackberry extract causes a catabolic response with increments in insulin sensitivity in rats.

Blackberry (Rubus sp.) fruit has a high content of anthocyanins, but its health benefits have not been sufficiently explored in healthy individuals. Thus, the aim of the study was to determine the effects of blackberry extract on lipid and glucose variables in female and male rats. Sprague Dawley rats were given a standard pellet (SD) or cafeteria (CD) diet supplemented (SD+R and CD+R) or not with Rubus extract for 80 days. Female rats given SD+R had lower body and liver weights than SD females; both sexes given SD+R showed lower plasma glucose and insulin, higher plasma NEFA, glycerol and 3-hydroxybutyrate, and higher liver concentration of triacylglycerols than SD rats. The homeostasis model of insulin resistance (HOMA) was lower in SD+R rats than in SD rats, but higher in CD rats. No effects of Rubus extract were observed in CD rats. In conclusion, Rubus extract, in rats given SD, decreased glycemia and increased insulin sensitivity. It also increased lipid breakdown in adipose tissue. The effects were greater in females than in males. No effect was seen in rats given CD, probably as a result of their high insulin resistance.

Method development for determination of (+)-catechin and (-)-epicatechin by micellar electrokinetic chromatography: annual characterization of field grown blackberries.

Berries are a rich source of antioxidants compounds, among which is the catechin group. Determination of the monomers (catechin and epicatechin) in fruits is a first step in the way to establish a relationship between polyphenols and their effects on human health. The purpose of this work is to develop a method to determine free catechins in blackberry by MEKC and to characterize levels of catechins in fresh fruits of Rubus fruticosus var. Lochness throughout the annual production period. A methanolic extract was prepared from fresh fruit. Then, it was evaporated and the residue was extracted with diethyl ether. MEKC conditions: phosphoric acid, 30 mmol/L; SDS, 40 mmol/L and triethylamine, 0.1% v/v at pH 2.3; -15 kV of voltage; 10-s hydrodynamic injection; 25°C temperature; and detection at 200 nm. Instrumental and interday precision were lower than 4.7 and 10% RSD, respectively. Only (-)-epicatechin was quantified in blackberries and ranged from 120 to 620 mg/kg fresh weight, which were the lowest values in December and the highest in June. A solid-liquid extraction and an MEKC method were successfully applied to determine (-)-epicatechins in blackberry for the first time. A strong dependence of (-)-epicatechin on the annual average temperature was observed.

Bacterial bioeffectors modify bioactive profile and increase isoflavone content in soybean sprouts (Glycine max var Osumi).

The effect of two bacterial strains to enhance bioactive contents (total phenolic compounds, total flavonoid compounds and isoflavones) and antioxidant activity on 3-day-old soybean sprouts were investigated. To identify bacterial determinants responsible for these effects, viable and UV-treated strains were delivered to wounded seeds at different concentration. Multivariate analysis performed with all the evaluated parameters indicated the different effectiveness of Stenotrophomonas maltophilia N5.18 and Pseudomonas fluorescens N21.4 based on different structural and metabolic determinants for each. N21.4 increased total phenolics and isoflavones from the genistein family, while N5.18 triggered biosynthesis of daidzein and genistein families coupled to a decrease in total phenolics, suggesting different molecular targets in the phenilpropanoid pathway. Only extracts from N5.18 treated seeds showed an improved antioxidant activity according to the ß-carotene bleaching prevention method. In summary, bioeffectors from both bacterial strains are effective tools to improve soybean sprouts quality; structural elicitors from N5.18 also enhanced antioxidant activity, being the best alternative for further development of a biotechnological procedure.

Physiological and Genetic Modifications Induced by Plant-Growth-Promoting Rhizobacteria (PGPR) in Tomato Plants under Moderate Water Stress.

Physiological, metabolic, and genetic changes produced by two plant growth promoting rhizobacteria (PGPR) Pseudomonas sp. (internal code of the laboratory: N 5.12 and N 21.24) inoculated in tomato plants subjected to moderate water stress (10% polyethylene glycol-6000; PEG) were studied. Photosynthesis efficiency, photosynthetic pigments, compatible osmolytes, reactive oxygen species (ROS) scavenging enzymes activities, oxidative stress level and expression of genes related to abscisic acid synthesis (ABA; 9-cis-epoxycarotenoid dioxygenase NCDE1 gene), proline synthesis (Pyrroline-5-carboxylate synthase P5CS gene), and plasma membrane ATPase (PM ATPase gene) were measured. Photosynthetic efficiency was compromised by PEG, but bacterial-inoculated plants reversed the effects: while N5.12 increased carbon fixation (37.5%) maintaining transpiration, N21.24 increased both (14.2% and 31%), negatively affecting stomatal closure, despite the enhanced expression of NCDE1 and plasma membrane ATPase genes, evidencing the activation of different adaptive mechanisms. Among all parameters evaluated, photosynthetic pigments and antioxidant enzymes guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) responded differently to both strains. N 5.12 increased photosynthetic pigments (70% chlorophyll a, 69% chlorophyll b, and 65% carotenoids), proline (33%), glycine betaine (4.3%), and phenolic compounds (21.5%) to a greater extent, thereby decreasing oxidative stress (12.5% in Malondialdehyde, MDA). Both bacteria have highly beneficial effects on tomato plants subjected to moderate water stress, improving their physiological state. The use of these bacteria in agricultural production systems could reduce the amount of water for agricultural irrigation without having a negative impact on food production.

Bacillus G7 improves adaptation to salt stress in Olea europaea L. plantlets, enhancing water use efficiency and preventing oxidative stress.

In addition to genetic adaptative mechanisms, plants retrieve additional help from the surrounding microbiome, especially beneficial bacterial strains (PGPB) that contribute to plant fitness by modulating plant physiology to fine-tune adaptation to environmental changes. The aim of this study was to determine the mechanisms by which the PGPB Bacillus G7 stimulates the adaptive mechanisms of Olea europaea plantlets to high-salinity conditions, exploring changes at the physiological, metabolic and gene expression levels. On the one hand, G7 prevented photosynthetic imbalance under saline stress, increasing the maximum photosynthetic efficiency of photosystem II (Fv/Fm) and energy dissipation (NPQ) and protecting against photooxidative stress. On the other hand, despite the decrease in effective PSII quantum yield (ΦPSII), net carbon fixation was significantly improved, resulting in significant increases in osmolytes and antioxidants, suggesting an improvement in the use of absorbed energy. Water use efficiency (WUE) was significantly improved. Strong genetic reprogramming was evidenced by the transcriptome that revealed involvement of the ABA-mediated pathway based on upregulation of ABA synthesis- and ABA-sensing-related genes together with a strong downregulation of the PLC2 phosphatase family, repressors of ABA-response elements and upregulation of ion homeostasis-related genes. The ion homeostasis response was activated faster in G7-treated plants, as suggested by qPCR data. All these results reveal the multitargeted improvement of plant metabolism under salt stress by Bacillus G7, which allows growth under water limitation conditions, an excellent trait to develop biofertilizers for agriculture under harsh conditions supporting the use of biofertilizers among the new farming practices to meet the increasing demand for food.

Lipo-Chitooligosaccharides (LCOs) as Elicitors of the Enzymatic Activities Related to ROS Scavenging to Alleviate Oxidative Stress Generated in Tomato Plants under Stress by UV-B Radiation.

Exposure to ultraviolet-B (UV-B) radiation can lead to oxidative damage in plants, increasing reactive oxygen species (ROS) production. To overcome ROS burst, plants have antioxidant mechanisms related to ROS scavenging which can be improved by elicitation with biological agents or derived molecules (elicitors), as they can trigger a physiological alert state called "priming". This work describes the effects of lipo-chitooligosaccharides (LCOs) treatment applied to tomato plants under UV-B stress. The LCOs used in the study are produced by three species of the genus Ensifer (formerly Sinorhizobium) (SinCEU-1, SinCEU-2, and SinCEU-3) were assayed on tomato plants under UV-B stress. LCOs were able to significantly increase most of the enzymatic activities related to ROS scavenging while non-enzymatic antioxidants were not modified. This response was associated with a lower oxidative stress, according to malondialdehyde (MDA) levels and the higher antioxidant capacity of the plants. Furthermore, the photosynthetic efficiency of LCOs-treated plants indicated a better physiological state than the control plants. Therefore, although more studies and deepening of certain aspects are necessary, LCOs have shown great potential to protect plants from high UV-B radiation conditions.

Bacillus H47 triggers Olea europaea metabolism activating DOXP and shikimate pathways simultaneously and modifying leaf extracts' antihypertensive activity.

Improvement of plant adaptation by beneficial bacteria (PGPB) may be achieved by triggering multiple pathways to overcome the environmental stress on plant's growth cycle, activating plant's metabolism. The present work reports the differential ability of three Bacillus strains to trigger olive tree metabolism, among which, only H47 was outstanding increasing iridoid and flavonol concentration. One-year old olive seedlings grown open air, under harsh conditions of water shortage in saline soils, were root-inoculated with three Bacillus PGPB strains throughout a 12-month period after which, photosynthesis was determined; photosynthetic pigments and bioactive secondary metabolites (iridoids and flavonols) were analyzed, and a study of gene expression of both pathways involved was undertaken to unravel molecular targets involved in the activation. All three strains increased plant fitness based on photosynthetic values, increasing energy dissipation capacity to lower oxidative stress; only H47 increased CO2 fixation and transpiration. Bacillus H47 was found to trigger synthases in the DOXP pathway (up to 5-fold in DOXP-synthase, 3.5-fold in Iridoid synthase, and 2-fold in secologanin synthase) associated to a concomitant increase in iridoids (up to 5-fold in oleuropein and 2-fold in its precursor secologanin). However, despite the 2-fold increases detected in the two predominant flavonols, gene expression was not enhanced, suggesting involvement of a pulse activation model proposed for innate immunity. Furthermore, the activity of leaf extracts to inhibit Angiotensin Converting Enzyme was evaluated, to explore further uses of plant debris with higher added value. Despite the increases in iridoids, leaf extracts from H47 did not increase ACE inhibition, and still, increased antihypertensive potential in oil obtained with this strain is to be explored, as leaves are the source for these metabolites which further translocate to fruits. In summary, Bacillus H47 is an effective strain to increase plant adaptation to dry and saline environments, activates photosynthesis and secondary metabolism in olive tree.

Modulation of Photosynthesis and ROS Scavenging Response by Beneficial Bacteria in Olea europaea Plantlets under Salt Stress Conditions.

Climate change consequences for agriculture involve an increase of saline soils which results in lower crop yields due to increased oxidative stress in plants. The present study reports the use of Plant Growth Promoting Bacteria (PGPB) as a tool to modulate plant innate mechanisms of adaptation to water stress (salinity and drought) in one year-old olive plantlets var. Arbosana and Arbequina. Integration of external changes in plants involve changes in Reactive Oxygen Species (ROS) that behave as signals to trigger plant adaptative mechanisms; however, they become toxic in high concentrations. For this reason, plants are endowed with antioxidant systems to keep ROS under control. So, the working hypothesis is that specific beneficial strains will induce a systemic response able to modulate oxidative stress and improve plant adaptation to water stress. Ten strains were assayed, evaluating changes in photosynthesis, pigments, ROS scavenging enzymes and antioxidant molecules, osmolytes and malondialdehyde, as oxidative stress marker. Photosynthesis and photosynthetic pigments were the most affected variables. Despite the specific response of each variety, the favorite targets of PGPBs to improve plant fitness were photosynthetic pigments and the antioxidant pools of glutathione and ascorbate. Our results show the potential of PGPBs to improve plant fitness modulating oxidative stress.

Tomato Bio-Protection Induced by Pseudomonas fluorescens N21.4 Involves ROS Scavenging Enzymes and PRs, without Compromising Plant Growth.

AIMS: to discover the interrelationship between growth, protection and photosynthesis induced by Pseudomonas fluorescens N21.4 in tomato (Lycopersicum sculentum) challenged with the leaf pathogen Xanthomonas campestris, and to define its priming fingerprint. METHODS: Photosynthesis was determined by fluorescence; plant protection was evaluated by relative disease incidence, enzyme activities by specific colorimetric assays and gene expression by qPCR. Changes in Reactive Oxygen Species (ROS) scavenging cycle enzymes and pathogenesis related protein activity and expression were determined as metabolic and genetic markers of induction of systemic resistance. RESULTS: N21.4 significantly protected plants and increased dry weight. Growth increase is supported by significant increases in photochemical quenching together with significant decreases in energy dissipation (Non-Photochemical Quenching, NPQ). Protection was associated with changes in ROS scavenging cycle enzymes, which were significantly increased on N21.4 + pathogen challenged plants, supporting the priming effect. Superoxide Dismutase (SOD) was a good indicator of biotic stress, showing similar levels in pathogen- and N21.4-treated plants. Similarly, the activity of defense-related enzymes, ß-1,3-glucanase and chitinase significantly increased in post-pathogen challenge state; changes in gene expression were not coupled to activity. CONCLUSIONS: protection does not compromise plant growth; N21.4 priming fingerprint is defined by enhanced photochemical quenching and decreased energy dissipation, enhanced chlorophylls, primed ROS scavenging cycle enzyme activity, and glucanase and chitinase activity.

Pseudomonas palmensis sp. nov., a Novel Bacterium Isolated From Nicotiana glauca Microbiome: Draft Genome Analysis and Biological Potential for Agriculture.

A novel Pseudomonas, designated strain BBB001T, an aerobic, rod-shaped bacterium, was isolated from the rhizosphere of Nicotiana glauca in Las Palmas Gran Canaria, Spain. Genomic analysis revealed that it could not be assigned to any known species of Pseudomonas, so the name Pseudomonas palmensis sp. nov. was proposed. A 16S rRNA gene phylogenetic analysis suggested affiliation to the Pseudomonas brassicae group, being P. brassicae MAFF212427 T the closest related type strain. Upon genomic comparisons of both strains, all values were below thresholds established for differentiation: average nucleotide identity (ANI, 88.29%), average amino acid identity (AAI, 84.53%), digital DNA-DNA hybridization (dDDH, 35.4%), and TETRA values (0.98). When comparing complete genomes, a total of 96 genes present exclusively in BBB001T were identified, 80 of which appear associated with specific subsystems. Phenotypic analysis has shown its ability to assimilate glucose, potassium gluconate, capric acid malate, trisodium citrate, and phenylacetic acid; it was oxidase positive. It is able to produce auxins and siderophores in vitro; its metabolic profile based on BIOLOG Eco has shown a high catabolic capacity. The major fatty acids accounting for 81.17% of the total fatty acids were as follows: C16:0 (33.29%), summed feature 3 (22.80%) comprising C16:1 ω7c and C16:1 ω6c, summed feature 8 (13.66%) comprising C18:1 ω7c, and C18:1ω6c and C17:0 cyclo (11.42%). The ability of this strain to improve plant fitness was tested on tomato and olive trees, demonstrating a great potential for agriculture as it is able to trigger herbaceous and woody species. First, it was able to improve iron nutrition and growth on iron-starved tomatoes, demonstrating its nutrient mobilization capacity; this effect is related to its unique genes related to iron metabolism. Second, it increased olive and oil yield up to 30% on intensive olive orchards under water-limiting conditions, demonstrating its capacity to improve adaptation to adverse conditions. Results from genomic analysis together with differences in phenotypic features and chemotaxonomic analysis support the proposal of strain BBB001T (=LMG 31775T = NCTC 14418T) as the type strain of a novel species for which the name P. palmensis sp. nov is proposed.

Bioeffectors as Biotechnological Tools to Boost Plant Innate Immunity: Signal Transduction Pathways Involved.

The use of beneficial rhizobacteria (bioeffectors) and their derived metabolic elicitors are efficient biotechnological alternatives in plant immune system elicitation. This work aimed to check the ability of 25 bacterial strains isolated from the rhizosphere of Nicotiana glauca, and selected for their biochemical traits from a group of 175, to trigger the innate immune system of Arabidopsis thaliana seedlings against the pathogen Pseudomonas syringae pv. tomato DC3000. The five strains more effective in preventing pathogen infection were used to elucidate signal transduction pathways involved in the plant immune response by studying the differential expression of Salicylic acid and Jasmonic acid/Ethylene pathway marker genes. Some strains stimulated both pathways, while others stimulated either one or the other. The metabolic elicitors of two strains, chosen for the differential expression results of the genes studied, were extracted using n-hexane, ethyl acetate, and n-butanol, and their capacity to mimic bacterial effect to trigger the plant immune system was studied. N-hexane and ethyl acetate were the most effective fractions against the pathogen in both strains, achieving similar protection rates although gene expression responses were different from that obtained by the bacteria. These results open an amount of biotechnological possibilities to develop biological products for agriculture.

Elicitation with Bacillus QV15 reveals a pivotal role of F3H on flavonoid metabolism improving adaptation to biotic stress in blackberry.

The aim of this study is to determine the involvement of the flavonol-anthocyanin pathway on plant adaptation to biotic stress using the B.amyloliquefaciens QV15 to trigger blackberry metabolism and identify target genes to improve plant fitness and fruit quality. To achieve this goal, field-grown blackberries were root-inoculated with QV15 along its growth cycle. At fruiting, a transcriptomic analysis by RNA-Seq was performed on leaves and fruits of treated and non-treated field-grown blackberries after a sustained mildew outbreak; expression of the regulating and core genes of the Flavonol-Anthocyanin pathway were analysed by qPCR and metabolomic profiles by UHPLC/ESI-qTOF-MS; plant protection was found to be up to 88%. Overexpression of step-controlling genes in leaves and fruits, associated to lower concentration of flavonols and anthocyanins in QV15-treated plants, together with a higher protection suggest a phytoanticipin role for flavonols in blackberry; kempferol-3-O-rutinoside concentration was strikingly high. Overexpression of RuF3H (Flavonol-3-hidroxylase) suggests a pivotal role in the coordination of committing steps in this pathway, controlling carbon flux towards the different sinks. Furthermore, this C demand is supported by an activation of the photosynthetic machinery, and boosted by a coordinated control of ROS into a sub-lethal range, and associated to enhanced protection to biotic stress.

Management of Plant Physiology with Beneficial Bacteria to Improve Leaf Bioactive Profiles and Plant Adaptation under Saline Stress in Olea europea L.

Global climate change has increased warming with a concomitant decrease in water availability and increased soil salinity, factors that compromise agronomic production. On the other hand, new agronomic developments using irrigation systems demand increasing amounts of water to achieve an increase in yields. Therefore, new challenges appear to improve plant fitness and yield, while limiting water supply for specific crops, particularly, olive trees. Plants have developed several innate mechanisms to overcome water shortage and the use of beneficial microorganisms to ameliorate symptoms appears as a challenging alternative. Our aim is to improve plant fitness with beneficial bacterial strains capable of triggering plant metabolism that targets several mechanisms simultaneously. Our secondary aim is to improve the content of molecules with bioactive effects to valorize pruning residues. To analyze bacterial effects on olive plantlets that are grown in saline soil, photosynthesis, photosynthetic pigments, osmolytes (proline and soluble sugars), and reactive oxygen species (ROS)-scavenging enzymes (superoxide dismutase-SOD and ascorbate peroxidase-APX) and molecules (phenols, flavonols, and oleuropein) were determined. We found photosynthetic pigments, antioxidant molecules, net photosynthesis, and water use efficiency to be the most affected parameters. Most strains decreased pigments and increased osmolytes and phenols, and only one strain increased the antihypertensive molecule oleuropein. All strains increased net photosynthesis, but only three increased water use efficiency. In conclusion, among the ten strains, three improved water use efficiency and one increased values of pruning residues.

A novel strategy for rapid screening of the complex triterpene saponin mixture present in the methanolic extract of blackberry leaves (Rubus cv. Loch Ness) by UHPLC/QTOF-MS.

Triterpene saponins are important chemical constituents in blackberry leaves and have significant antimicrobial activity, among other healthful properties. In this study, a new UHPLC-MS method was optimized with outstanding efficiency for a non-targeted metabolic approach and comprehensive analysis of bioactive compounds in blackberry leaves (Rubus cv. Loch Ness). With minimum sample treatment, phenols and triterpene saponins, among others, were separated according to their elution times. Once separated, the major triterpene saponins were classified by their aglycone moieties and their structures tentatively identified based on their accurate mass spectra in positive and negative ESI mode. By the use of UHPLC coupled to a TOF, a high-resolution and accuracy mass analyzer, reliable molecular formulas of the detected compounds were predicted, and along with the generated MS spectra in full scan mode, allowed the tentative identification and classification of the most abundant triterpene saponins presented the samples analyzed. A rapid and comprehensive strategy to study the complex saponin profile is presented. Unlike other LC-MS/MS methods, the proposed method requires just one mass analyzer, and to our knowledge, this is the first systematic study on triterpene saponins in blackberry leaves.

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria.

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.

The inhibitory potential of Montmorency tart cherry on key enzymes relevant to type 2 diabetes and cardiovascular disease.

The inhibitory potential of Montmorency tart cherry on glycemia regulation and other enzymes relevant to inflammation were evaluated. Tart cherry has superior inhibitory potential against key enzymes associated with carbohydrate digestion linked to hypertension. In particular, α-amylase activity was significantly inhibited (IC50 = 3.46 ±â€¯0.06 mg/ml), whereas we observed mild inhibition of α-glucosidase (IC50 = 11.64 ±â€¯0.65 mg/ml). Angiotensin I-converting enzyme inhibition was also strong by about 89%. Tart cherry extract showed strong to moderate inhibitions of cyclooxygenase-1 (65%), lipoxygenase (64%), cyclooxygenase-2 (38%) and xanthine oxidase (26%), respectively. Anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, were strong inhibitors of α-amylase and α-glucosidase. Kaempferol showed relatively potent inhibition on COX and XO. It was revealed that some pairs of metabolites manifest positive or negative interactions against XO enzyme inhibition. Inhibition of all these enzymes provides a strong biochemical basis for management of type 2 diabetes and cardiovascular disease by controlling glucose absorption, reducing associated hypertension and inflammation.

Transcriptomics, Targeted Metabolomics and Gene Expression of Blackberry Leaves and Fruits Indicate Flavonoid Metabolic Flux from Leaf to Red Fruit.

Blackberries (Rubus spp.) are among the high added value food products relevant for human health due to the increasing evidence of the beneficial effects of polyphenols, which are very abundant in these fruits. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress. Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a de novo transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs. Transcripts were aligned against Fragaria vesca genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20,463 genes common to leaves and fruits, while 6,604 genes were significantly overexpressed only in fruits, while another 6,599 genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present. Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidin-3-glucoside as the only common compound identified. Next, RT-qPCR of the core genes in the flavonol anthocyanin pathway and regulatory MYB genes were carried out. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels. On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis upon ripening. This suggests that, in addition to the biosynthesis taking place in the fruits during ripening, translocation of flavonols from leaves to fruits contributes to the high amounts of bioactives starting to accumulate in fruits.